The Gram-negative bacterium, Rhizobium meliloti, and its eukaryotic host, alfalfa, interact symbiotically in developmental sequence to form nitrogen fixing root nodules. In addition to its great economic importance, this process offers unique opportunities to study cell-cell interactions using genetics and biochemical techniques. I plan to carry out studies on individual bacterial genes involved in early stages of the developmental sequence such as recognition, invasion, and stimulatioof host cell division. If have identified R. meliloti mutants which affect these early stages, and have obtained cloned fragments of wil-type R. meliloti DNA which complement the mutant phenotype. I will identify and clone other genes as part of this project. The stage at which the genes are first requied is identified through mutant phenotypes. Molecular level analyses of these genes can thus be tied in with experimental studies of the developmental sequence. I will test whether the genes controlling early steps in the symbiosis are expressed constitutively, or require certain physiological or nutritional conditions and/or the presence of the plant host for activaton. Gene expression will be followed using (a) cloned DNA sequences from nodulation genes to follow RNA populations in bcterial cells; and (b) antibodies made against proteins expressed from cloned nodulation genes in mini-cell synthesis systems. Coding regions in nodulation genes will be mapped by site-specific transposon mutagenesis of cloned genes. Mini-cell expression of these Tn5-mutated fragments will be used to analyze transcription units, revealing the number and extent of products from those genes. If any genes are found to be influenced by the host, then control of their expression will be studied using in vitro fusions of nod promoters to lac or drug-resistance structural genes. This project will determine the location and size of early symbiotic genes, identify gene products and test the time of their expression. A potential future benefit from this research will be that the cloned nodulation genes, urified gene products and antibodies aised against them will be useful as tools to locate the site of action of these products, and to probe individual early events of the symbiosis.